Animal cell colony picking apparatus and method

ABSTRACT

An apparatus and method for automated picking of animal cell colonies. A picking head comprising a plurality of hollow pins is provided. The apparatus has an integrated imager for capturing an image of adherent or non-adherent animal cell colonies held in liquid or semi-solid medium. Image processing identifies the locations of the animal cell colonies to be picked. Picking an animal cell colony is performed by aligning each of the hollow pins in turn with a target animal cell colony location, introducing the hollow pin into the medium, and aspirating the animal cell colony into the hollow pin. In the case of an adherent colony, the distal end of the pin is forced into oscillation to detach the animal cell colony prior to aspiration. The animal cell colony is dispensed into a well plate by increasing pressure in the fluid conduit.

BACKGROUND OF THE INVENTION

The invention relates to an apparatus and method for picking mammalianand other animal cell colonies.

Automated apparatus for a variety of repetitive tasks relating togenomics and proteomics have become widespread. For example, automatedapparatus have been commercially available for several years to performmicroarraying, bacterial colony picking and gel coring [1-5]. However,an apparatus for automated picking of mammalian cell colonies has notbeen available.

Mammalian cell colonies are usually provided in a sample in one of twotypes. In the first type, the mammalian cell colony is held insuspension in a semi-solid medium. In the second type, the mammaliancell colony is adhered to a substrate and immersed in a liquid medium.

A problem in automating the mammalian cell colony picking arises for thefirst type in locating, removing and dispensing individual target cellcolonies from the semi-solid medium. For adherent colonies, there is theadditional problem that the colony needs to be detached from thesubstrate before it can be removed. The standard (non-automated)practices for detaching adherent colonies are by scraping with ascalpel, or other mechanical blade, and digestion usually with trypsin.Neither of these practices is well suited to automation.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided anapparatus for picking animal cell colonies comprising: an apparatus bedfor arranging a sample container comprising a plurality of animal cellcolonies held in a medium; a camera for capturing images of the animalcell colonies; image processing software for identifying animal cellcolony locations from captured images; and a picking head movable aroundthe apparatus bed using positioning motors to animal cell colonylocations identified by the image processing software, wherein thepicking head comprises a plurality of hollow pins connected throughfluid conduits to a pressure controller that is operable to aspiratequantities of medium from the sample container into the hollow pins, toretain the medium and to expel it when required, thereby allowing animalcell colonies to be picked from the medium.

According to a second aspect of the invention there is provided a methodfor automated picking of animal cell colonies using an apparatus,preferably according to the first aspect of the invention, comprising:

-   -   a) providing a picking head comprising a plurality of hollow        pins, the picking head being movable over the apparatus using        positioning motors;    -   b) placing a sample container including a plurality of animal        cell colonies held in a medium onto the apparatus, and also a        dispensing container;    -   c) using machine vision and image processing to identify animal        cell colony locations in the sample;    -   d) moving the picking head to above the sample container;    -   e) picking an animal cell colony by aligning one of the hollow        pins with one of the animal cell colony locations, introducing a        distal end of the hollow pin into the medium, and aspirating the        animal cell colony at that location into the hollow pin; and    -   f) dispensing the picked animal cell colony by moving the        picking head to above the dispensing container and expelling the        picked animal cell colony into the dispensing container.

The automated process can be used to sort or pick animal cell colonieswhich comprise, express or secrete a biological molecule, such as aprotein or carbohydrate of interest, that may be detected using cellimaging. The animal cell colonies may be detected optically in a varietyof ways, for example with the aid of fluorescence stains,non-fluorescent stains, or without any stains; by using Ramanscattering, by using phase contrast or Nomaski interference imaging.

The colonies will most usually be mammalian cell colonies, but otheranimal cell colonies, such as insect cell colonies could also be picked.The target animal cell colonies can be discriminated on the basis of avariety of attributes, such as shape, size, color or molecular contentthat may be within the cell, in the membrane or secreted, or by anycombination of multiple attributes.

The picking step may comprise repeating the aligning and aspiratingsteps for multiple ones of the hollow pins to pick multiple ones of theanimal cell colonies.

The dispensing container may comprise an array of wells separated by acharacteristic spacing in which case it is preferred that the hollowpins are also arranged with the characteristic spacing so that theexpelling step can be performed in parallel for all the hollow pins. Itis convenient if the hollow pins are arranged in a characteristicspacing matched to a well plate standard spacing in order to reduce headmotion and also to allow the process to be parallelized.

The animal cell colonies may be contacted with or express a fluorescentprotein (FP), e.g. green fluorescent protein (GFP), to assist the imageprocessing. The FP may be within the cells, on the surface of the cells,or secreted into the medium surrounding the colony. The process can bebased on any molecule that is detectable with an antibody, ligand orreceptor.

The picking head can advantageously further comprise a drive mechanismfor causing lateral oscillation of distal ends of the pins to facilitatedetachment of adherent animal cell colonies. For example, the drivemechanism can be configured to cause rotary motion of the distal ends ofthe pins. Other types of motion, such as linear, would also be possible.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the invention and to show how the same maybe carried into effect reference is now made by way of example to theaccompanying drawing which show:

FIG. 1 is a perspective view of the lower part of an apparatus embodyingthe invention;

FIG. 2 is a perspective view of the apparatus;

FIG. 3 shows features of the light table illumination system of theapparatus;

FIG. 4 is a schematic side view showing the optical design of theapparatus;

FIG. 5 is a perspective view of the head of the apparatus;

FIG. 6 is a perspective view of one pin of the head with associatedagitation motor;

FIG. 7 is a schematic section of the pin and motor of FIG. 6 in use;

FIG. 8 is a series of schematic captions illustrating the main steps inpicking a an adherent animal cell colony;

FIG. 9 is a schematic drawing of the fluidics elements of the apparatus;

FIG. 10 is a block schematic diagram showing the control system of theapparatus;

FIG. 11 is a flow diagram showing an example process carried out by theapparatus; and

FIG. 12 is a flow diagram of the cell colony picking part of the processof FIG. 11.

DETAILED DESCRIPTION

FIG. 1 is a perspective view of the lower part of an apparatus embodyingthe invention. The upper part is not shown in order to reveal the mainbed 10 of the apparatus. A light table plate 12 made of a translucentmaterial, known as opal acrylic, is mounted flush with the main bed. Thelight table plate could be made of other translucent acrylic or glassmaterial, for example shot-blasted glass or perspex.

A set of pegs or clamps 14 (four in the figure) is provided around theperiphery of the light table plate 12 for locating and securingbiological sample containers containing cell colonies, such as Petridishes 13, on the light table. The colonies could also be provided inQ-trays, omni-trays or any other suitable container. The light tableplate 12 is illuminated from below by optical equipment accommodatedunder the main bed in the space provided in the apparatus's main base16.

The animal cell colonies may be human cell colonies or other mammaliancell colonies, or insect cell colonies. The cells may be immortal,embryonic or stem cells, for example. Typically, the cell colonies willbe grown in tissue culture.

The apparatus has a mammalian cell colony picking head 18 which ismovable over the main bed of the apparatus by x- y- and z-positioners20, 22 and 24 respectively. The head illustrated comprises an array ofhollow pins 26, each connected to a fluid line. In one example, the headhas a 1×8 array of pins with each pin 26 having its own fluid line 28for aspiration (sucking) and dispensing (expulsion) of a cell colony toperform cell colony picking from a container followed by deposition ofthe cell colony sample into a well of a well plate 29 or other targetlocation. It will be appreciated that multiple well plates 29 willnormally be used, although only one is illustrated for the sake ofsimplicity. A grid of well plate receiving bays can be provided on theapparatus main bed. Automation of well plate handling may also beprovided, for example using a “hotel” system, or a fully automated wellplate feeder and stacker, as is known in the prior art. The head 18 iscarried by the z-positioner, which is in turn carried by they-positioner, which is carried by the x-positioner. The head also hasmounted on one side a camera 19 for machine vision. The camera 19 mayinclude a zoom objective lens, or swappable objective lenses, in orderto provide different magnifications, and hence resolutions.Alternatively, a further head-mounted camera (not shown) may be providedso that there are two cameras with different objective magnifications,one for low resolution, large area views and the other for small area,high resolution views. In use, the low resolution camera is used formapping over the whole Petri dish or other container to identify thecolony locations, and then the high resolution camera is used forimaging each colony. It will be understood that a similar procedure canbe carried out with a single head-mounted camera with variablemagnification.

Adjacent the head 18 the z-positioner may also have attached thereto awell plate gripper (not shown) to allow well plates to be moved aroundthe main bed of the apparatus.

A single well plate 29 is illustrated on the main bed of the apparatusby way of example. The main bed may be provided with stations for wellplates and colony dishes of various standard types.

After picked cell colonies have been dispensed into their wells, thepins need to be cleaned before they can be used to pick up further cellcolonies so as to avoid cross-contamination. For the pin cleaning, awashing and drying station 2 is used. The washing part includes firstand second baths 4 and 6. The drying part 8 consists of a cavity forblowing air onto the pins and preferably also halogen or other lamps.The lamps serve to sterilize and dry the pins by heating. The blower canassist drying, but if lamps are provided, serve principally to cool thepins after heating.

After completion of a run, the x- y- and z-positioners 18, 20 and 22move the head over to the washing and drying station 2. The pins arelowered and dipped into the first wash bath 4, containing water, forexample, or a water/bleach mixture. The first bath 4 may be fitted withupstanding brushes immersed in the cleaning liquid, in which case the x-and y-positioners are used to move the pins over the brushes in a rotarymotion in the xy-plane. The pins are also preferably purged with asterilizing solution, such as bleach. The head is then moved to thesecond bath 6, which contains, for example, ethanol for more cleaning.Ethanol is used in the last bath 6 in view of its volatility whichassists the subsequent drying of the pins. It will be appreciated thatthe named cleansing agents are mentioned to give concrete examples.Other cleansing agents are sometimes used.

The head is then moved over to above the drying station 8. If provided,the halogen lamps are switched on at this point to heat the pins. An airblower is then switched on to dry the pins and/or to cool them toambient temperature if they have been heated by the halogen lamp.

The main bed may also include other standard equipment such as a wastechute, a well plate delidder, well plate shakers, or well plate hotel.None of these is illustrated. The apparatus may also be provided with anautomated well plate feeding and stacking mechanism and an automatedcell colony dish feeding and stacking mechanism. Neither is shown.

FIG. 2 is a perspective view of the apparatus embodying the invention.In comparison to FIG. 1, the apparatus also shows the upper part of themachine. The upper part is principally formed of a light-tight andgas-tight cover 30 with two sides 32, a back 34 and a roof 36. Aso-called high-efficiency particulate air (HEPA) filter unit 35 is alsomounted on the roof 36. The working volume of the apparatus contained bythe main bed 10 and the cover 30 can be kept in a controlled environmentsubstantially free of contaminant particles by supplying filtered airinto the working volume through the HEPA filter unit 35 and maintainingthe working volume at slight overpressure so that unfiltered air fromthe surroundings is prevented from entering the working volume.

On the front side of the machine there is a hinged door 38 to provideaccess. Mounted in the roof, there is a detector unit 40 housing anarray detector, in the form of a CCD chip, and associated imagingoptics, in the form of an objective lens and an appropriate filter. Theimaging optics are designed to have the upper surface of the light tableplate as the imaging plane, or a slightly higher plane to take accountof the usual thickness of the cell colony dish base. The CCD chip can becooled with a Peltier cooler (not shown) to reduce noise. If a loweroperating temperature is desired to further reduce noise, a cryogen suchas liquid nitrogen could be used, for example with a closed cyclecryostat. The array detector need not be a CCD and could be any suitabletwo-dimensional array detector, such as a multi-channel plate (MCP). Thearray detector may be cooled, for example with a Peltier device or usinga cryogen such as liquid nitrogen.

The detector unit 40 is contained in a cylindrical housing boltedupright on the roof of the machine so that the principal optical axis“O” of the detector unit is perpendicular to the plane of the main bedof the apparatus. Optical access to the light table below is provided byan aperture in the roof.

FIG. 3 shows features of the light table illumination system of theapparatus embodying the invention. The figure is a perspective view fromone side of the machine from a position looking down onto and across theplane of the main bed of the machine, but with the main bed not shown.The plane of the main bed (and the light table plate) is marked withreference numeral 10. The main part of the illumination system is asource unit 50 which is rectangular in footprint having dimensionsapproximately matching those of the light table illumination plate (e.g.about 300×200 mm) and which has a thickness of approximately 20 mm.Arranged in the base of the source unit is a plurality of blue lightemitting diodes (LEDs) that emit at a nominal center wavelength of 473μm. (In other embodiments different color LEDs could be used.) The LEDsare arranged in four banks 52 (dashed lines) with each bank being apackaged unit of 100 surface mounted LEDs with an integrated Fresnellens. Alternatively, discrete LEDs could be used spread over the area ofthe source unit. Arranged over the LEDs covering the whole area of thesource unit is a sheet of filter (not shown) followed by a sheet ofholographic diffuser 54. The holographic diffuser is a sheet of plasticmaterial with a microsculpted surface relief structure made by anembossing process using a holographically produced blank (see e.g. U.S.Pat. No. 5,534,386: Physical Optics Corporation [6]). The diffuserhomogenizes the LED light output, so that the intensity distribution oflight from the LEDs on the light table plate is equalized. Arranged oneither side of the source unit there are strip light units 56 forproviding white light illumination to the light table plate for contrastimaging.

FIG. 4 is a schematic side view showing the optical design. At the topof the figure, the detector unit 40 can be seen mounted on the roof 36.The detector unit 40 contains a CCD chip 42 with associated collectionoptics 44 illustrated schematically as a single objective lens. It willbe appreciated that any appropriate lens (or mirror) combination may beused in order to image the light table area onto the active surface ofthe CCD chip. The optical axis “O” of the detector unit 40 is alsoshown. The detector unit 40 also includes a filter 46. This is abandpass filter for filtering out the LED output. A 620 nm centerwavelength with 35 nm bandpass is selected for the blue LEDs mentionedabove. It will be appreciated that in general an appropriate selectionof bandpass or cutoff filter will be made having regard to the outputresponse of the LEDs, and the excitation and emission bands of thefluorescent stain to be used. Moreover, in some applications, forexample with contrast imaging, a filter may be dispensed with. In themiddle of the figure, the light table plate 12 and dish clamps 14 areillustrated, with the light table plate 12 lying generally in the planeof the apparatus main bed 10. At the bottom of the figure, the lightsource unit 50 is shown with its LEDs 52, filter 53 and diffuser 54. Thefilter 53 is a bulk dyed band pass filter of the kind used fortheatrical lighting which is blue in the present example where blue LEDsare used. The filter 53 is effective for removing undesired componentsof the LED output. Specifically in the case of blue LEDs it has beenfound that a small proportion of the LEDs malfunction by emittingwavelength components outside the blue into the green and red. Adifferent color filter can be chosen depending on the output wavelengthsof the LEDs used. The angled strip light units 56 are also evident.

A CCD exposure time of around 10 seconds to 3 minutes is usuallysufficient to image cell colonies. The exposure time will depend on anumber of factors, for example the type of stain used. One useful stainis trypan blue. The exposure time is proportional to illuminationintensity, so that exposure time can be reduced by using more intenseillumination from white light sources, lasers or LEDs.

FIG. 5 is a perspective view showing a part of the head 18 in moredetail. The end portions of the hollow pins 26 are visible protrudingthrough the bottom of their housing parts. The hollow interior of thepins extends upwards and then through a right angle bend to emerge atstubs 27 for connecting flexible fluid lines (not shown in FIG. 5, butsketched in FIG. 1). It is also noted that the head 18 with pins can beremoved as a single piece and autoclaved for sterilization.

Automated head swapping could also be provided as described in U.S. Ser.No. 10/144,763 by Ruddock, the contents of which is incorporated hereinby reference. Various multiple head configurations are contemplated. Forexample, to improve speed, two heads similar to that shown in FIG. 5could be provided. While one of these heads is being used, the other canbe cleaned in an automated wash/dry station, thus eliminating dead timethat would otherwise arise during head sterilization, as described inU.S. Ser. No. 10/298,948 by Elverd, Haslam and Ruddock, the contents ofwhich is incorporated herein by reference. Another possibility is toprovide a specialist imaging head for carrying the camera (or cameras).By providing a separate imaging head, this reduces the weight of theheads, thereby increasing the speed with which they can be scanned andavoiding overloading problems. This may be especially useful if theimaging head includes further parts, such as its own spectrometer ormultiple cameras.

FIG. 6 is a perspective view of one of the pins 26 in more detail. Aswell as the pin an electric motor 60 is also shown fitted alongside thepin 26. The hollow pin 26 comprises both outer and inner pins 62 and 64,with the inner pin 64 arranged coaxially inside the outer pin 62. Bothpins are made of stainless steel. The inner pin 62 forms the end of thefluid path that includes the flexible tubing. The electric motor 60 isconnected to the inner pin 64 by a connecting rod 66 that is driven by acrank mounted on the motor 60. The motor thus drives the inner pin 62 sothat it describes a rotary motion, with the motion being accommodated bybending of the inner pin 64.

In the present embodiment, the inner pin 64 has an inside diameter of0.7 mm an outside diameter of 1.07 mm. The outer pin 62 is 35 mm longand has a 5 mm outer diameter, tapering to 4.2 mm at its end, and a 3.2mm inner diameter. These dimensions are suitable for picking cellcolonies of average size circa 0.5 mm.

FIG. 7 is a schematic section of the pin 26 and motor 60 showing the pinin use for picking adherent cell colonies. The inner and outer pins 64and 62 can be seen, as well as the motor 60 and connecting rod 66. As isshown, the end of the inner pin 64 is recessed axially inside the end ofthe outer pin 62 by a distance ‘d1’. In the present embodiment values ofd1=0.25-0.5 mm have been used. Other values in the range 0.1 to 2 mmcould be suitable, depending on the relevant parameters such as averagecolony size and liquid viscosity. The figure shows the pin 26 lowered inposition for picking an adherent cell colony 74. The pin 26 is loweredso that the distal end of the outer pin 62 is immersed in the liquid 72and offset by a small amount ‘d2’ from the upper surface of the samplecontainer base (substrate) 76 on which the cell colonies are grown. Inthe present embodiment, d2 is varied between about zero (i.e. buttingagainst the plate 76) and 0.5 mm, although larger offsets could becontemplated, for example up to 2 mm. Values of zero (i.e. butting), 0.1mm or 0.2 mm are usual. The pin 26 is also aligned with the cell colony74 as determined by the xy coordinates of the colony determined by theimage of the sample taken with the camera 40 and subsequent imageprocessing.

In the position illustrated, the motor 60 is actuated to oscillate theend of the inner pin 64, thereby creating turbulence in the liquid 72.An oscillation frequency of around 100 Hz has been successfully used.Other frequencies would probably also work. The forces induced by thismotion have been found sufficient to detach the cell colony and allowaspiration of the detached cell colony into the hollow pin, which asmentioned above forms the end of a capillary 70. The inner pin 64 isconstrained by a flange 68 which fits into the top of the outer pin 62and has a central through hole through which the inner pin 64 passes ina push fit.

In the case of non-adherent colonies held in a semi-solid growth medium,it will be understood that the colonies are not attached to the plate sothere is no need to vibrate the pin.

FIG. 8 is a series of schematic captions A-E illustrating the main stepsin picking a targeted mammalian cell colony. In caption A, the pin 70 isbeing lowered over an adherent target colony 74 immersed in liquid 72after alignment of the xy-coordinates. In caption B, the pin tip isimmersed directly over the colony ready for carrying out the detachmentprocess. Caption C shows the situation after the vibration-induceddetachment in which aspiration is taking place. A column of liquid inwhich the colony is in suspension is drawn up into the pin by loweringof pressure in the pin. Caption D shows the situation after the pin hasbeen raised out of the sample container. The colony is retained by alower than atmospheric pressure being maintained in the pin. The headcan then be moved over to a well plate for dispensing. Caption E showsthe situation in which the end of the pin has been lowered into a wellof a well plate, and the colony ejected from the pin by raising thepressure in the pin, thereby completing the cell picking operation.

The above description has taken the example of an adherent colony. Itwill be understood that when a colony is in suspension, a simplifiedform of the same process can be carried out with the steps associatedwith detaching an adherent colony being omitted. It will also beunderstood that some of the parts of the apparatus are redundant in thecase of picking cell colonies from suspension and could be omitted ifthe machine did not need to have a capability for handling adherentcolonies. For example, the outer pin could be omitted as well as themotor and associated drive parts.

FIG. 9 is a schematic drawing of the fluidics elements of the apparatus.One of the pins 26 is shown connected to its fluid line 28 made offlexible tubing which leads to a manifold 81 mounted on the housing of afluidics unit 86. (The manifold 81 is used for receiving all the fluidlines 28, although only one is shown.) The manifold 81 also receives afurther fluid line 101 from a liquid supply vessel 103 through a fluidline 101 which accesses the liquid in the supply vessel 103 through asealed top flange 105. The liquid in the vessel 103 may be held underpressure. In the interior of the fluidics unit 86, the fluid supply isconnected from the manifold 81 through a fluid line 99 to a normallyopen (N.O.) port 93 of a valve 85, and the other side of the fluid pathleading to the pin 26 is connected from the manifold 81 through a fluidline 97 to a normally closed (N.C.) port 89 of the valve 85. Thefluidics unit 86 also houses a pump 83, which is a reciprocatingpiston/cylinder pump which connects through a fluid line 95 to a commonport (COM) 87 of the valve 85. The valve 85 and also the pump 83 areconnected to a fluidics control unit 84 by electrical control lines. Thefluidics control unit 84 is itself connected to and driven by a controlcomputer (not shown in this figure).

In use, the valve 85 is controlled as follows. When the valve 85 is inits rest state with no inputs, the N.O. port 93 is open and the N.C.port 89 is closed. This connects the reciprocating pump 83 to the fluidvessel 103 so that it can draw liquid out of the reservoir by suitabledownward motion of the pump piston in its cylinder. On the other hand,when the valve 85 is in its actuated state with an energizing inputsignal, the N.O. port 93 is closed and the N.C. port 89 is open. Thisconnects the reciprocating pump 83 to the pin 26 allowing the liquidcolumn in the fluid path formed by elements 26, 28, 97 and 95 to bemoved in either direction by motion of the pump cylinder. This providesthe fine control for the aspiration and expulsion of animal cellcolonies shown schematically in FIG. 8 as described above. Raising ofthe piston also allows purging of the fluid path during cleaning. Forcleaning, the fluid vessels can be swapped by hand. Alternatively, anautomated switching between different fluid vessels can be providedusing additional computer-controlled valves. On occasion, a pressurizedgas canister could be used as a fluid vessel (e.g. for compressed gascleaning), so the fluid vessels need not necessarily be liquidcontaining. It will be understood that the vessels 103 can be sharedamong multiple pins and there need not be a separate vessel 103 for eachpin. Individual pumps could also be shared between two or more valves toreduce cost.

FIG. 10 is a block schematic diagram showing the control system of theapparatus for coordinating the various components to perform theprocesses described above. A computer (PC 90) is used as the principalcontrol component and is connected by electronic links using standardinterfacing protocols to the various components that are part of theautomated control system. The control is effected by control software 91resident in the PC 90. Image processing software 92 is also resident inthe PC 90 and linked to the control software 91. The detector unit 40 inthe form of a CCD camera is connected to the PC 90 for receiving digitalimages captured by the camera. An illumination and filter controller 80is connected to the PC 90. This controller is used to control the LEDs52 and white light source unit 56 used to illuminate the light tableplate 12 and also to select appropriate filter 46 from a motor drivenfilter wheel. A washer/drier controller 82 is connected to the PC 90 andused to control the blower and the halogen lamps of the drier. Thepositioners 20, 22, 24 for moving the head 18 are connected to the PC90. The head-mounted camera 19 is connected to the PC 90 for receivingdigital images captured by the head-mounted camera 19. These are usedfor aligning the pins of the head with the various parts of the washingand drying station 4, 6, 8, well plates 29, colonies etc. The fluidlines 70 are connected to the fluidics unit 86 which is controlled bythe fluidics control unit 84 connected to the PC 90. The fluidicscontrol unit 84 is used to control the pressure in the fluid lines toallow aspiration, retention and expulsion of liquid from the sample. Thefluidics control unit 84 also controls the wash cycle of the pins andfluid lines, whereby cleaning fluid from the baths 4, 6 is aspirated andexpelled from the ends of the pins during the cleaning cycle.

FIG. 11 is a flow diagram showing an example process carried out by theapparatus. The process has the following steps:

-   -   S1 Place cell dishes in imaging area, i.e. on light table plate        12    -   S2 Illuminate imaging area 12 using either the colored light        source 52 (typically for detecting stained cell colonies using        fluorescence) or the white light source 56 (typically for        unstained colonies)    -   S3 Capture image(s) using CCD camera 40    -   S4 Perform software-based image analysis to detect cell colonies        to pick—creating a “pick list”    -   S5 Assign the robotic apparatus to collect cell colonies from        the pick list (described in more detail below)    -   S6 Store all images and data    -   S7 Remove well plates containing the picked colonies

Regarding steps S2 to S4, the cell colonies may be made visible usingfluorescence. For example, a fluorescently labeled antibody may be usedthat binds to a protein of interest secreted from, expressed on thesurface of, or contained within the cell colonies. During imaging of thefluorescence, the cell colonies appear as brightly emitting spots whenilluminated with an appropriate excitation wavelength (e.g. with blueLEDs). Depletion of the fluorescent antibody in the immediate vicinityof the cell colonies also occurs which has the effect of producing an“inverse halo”, i.e. a darker area around each bright colony which aidsthe image processing.

Alternatively, in the case of staining, the cell colonies are madevisible by admixing a suitable stain into the suspension. It is howevernoted that it is possible to visualize many colonies without staining orfluorescence.

FIG. 12 is a flow diagram showing in more detail Step S5, the cellcolony picking part of the process. Step S5 of the process has thefollowing sub-steps:

-   -   S5.1 Select pick list element for picking    -   S5.2        -   Move unused pin to above the assigned xy coordinate of the            cell colony to be picked        -   “Fire” (i.e. lower) pin to colony picking position with the            end of the pin introduced into the medium over the target            cell colony by offset ‘d2’        -   For adhered colonies only—agitate pin end to dislodge target            cell colony        -   Aspirate defined volume        -   Retract pin and retain sample while other pins are fired    -   S5.3 Unless all pins are used or all colonies in the pick list        have been picked, repeat Step S5.2    -   S5.4 Move the head over to the well plate and align the pins        with the wells—then dispense all the samples simultaneously into        the well (microtitre) plate    -   S5.5 Clean all the pins using the washer and drier station 2    -   S5.6 Unless all colonies in the pick list have been picked move        the head back to the sample area and return to Step S5.1

This completes Step S5.

This concludes the description of the main embodiment. Various possiblealternatives are now mentioned.

The aspiration has been described in terms of having a liquid columnthrough the fluid lines and into the pins. An alternative would be tomaintain a liquid column in the majority of each fluid line, but to havea plug of air or inert gas, such as helium or nitrogen, in the pin tip.Sample could be aspirated by drawing up the liquid column and air plugtogether, so that the air plug separates the sample liquid or semi-solidmedium from the main body of the liquid column. The use of a gas plugoffers improved sterility, since sample liquid is isolated from the mainliquid column by the gas plug.

Alternative optical configurations are possible. For example, a lasersource could be used, such as an ion laser, in place of light emittingdiodes. The laser beam could be scanned over the light plate's lowersurface. In this case a single channel detector could be used. Suitableion lasers include argon ion and krypton ion sources. It will beappreciated that the beam scanning can be performed with appropriatescanning mirror optics. Emission wavelengths over a wide range could beused, for example 200 nm to 1.6 μm, or outside this range.

The apparatus can be used with a variety of optical based methods.Simple contrast imaging can be used, or more sophisticated spectroscopicmethods based on absorbance, luminescence or Raman scattering. If moresophisticated spectral analysis is needed, such as for resonant Ramanscattering, the collection optics may include a spectrometer orcontinuously tunable bandpass filter placed in front of the detector. Inorder to achieve significant absorbance changes, very highconcentrations of dyes must be used and many cells are needed to achievesignificant changes in optical density. Preferably, the optical basedmethods rely on fluorescence and/or luminescence which are moresensitive assay methods compared to absorbance.

It is also noted that although the main embodiment shows illumination ofa light table from below, illumination from above could be used eitherinstead of or in combination with illumination from below.

In some cases, the head-mounted camera (or cameras) could be usedexclusively and the roof-mounted camera could then be dispensed with.

Luminescent substrates include, but are not limited to, luciferase,luciferin (Anal Biochem (1994) 219, 169-181)m aequorin (Methods Enzymol.(1978) 57, 271-291), and alkaline phosphatase (Clin. Chem (1996) 42,1542-1546).

Fluorescent substrates can yield a very bright signal, which enablesthem to be easily detected at the single cell level. A preferredfluorescent protein is green fluorescent protein—such as a modified(e.g. mutated) GFP. By way of example only, GFP which may conjugated toone or more antibodies for the detection of a protein of interest.

The GFP of the jellyfish Aequorea victoria is a protein with anexcitation maximum at 395 nm and an emission maximum at 510 nm and doesnot require an exogenous factor. The properties and uses of GFP for thestudy of gene expression and protein localization have been discussedin, for example, Nat Cell Biol (2002) 4, E15-20; Biochemistry (1974) 13,2656-2662; Photochem. Photobiol. (1980) 31, 611-615; Science (1994) 263,12501-12504; Curr. Biology (1995) 5, 635-642; and U.S. Pat. No.5,491,084.

Accordingly, the apparatus of the present invention may be used withdifferent fluorescent and non-fluorescent proteins and stains.

As further light emitting diode types become commercially available(e.g. in the ultraviolet), more groups can be provided so that theapparatus can be developed to provide a greater range of possibleexcitation wavelengths. The apparatus can also be made multi-functionalby providing light emitting diodes of different types, for example afirst group for outputting in a first wavelength band (e.g. blue) and asecond group for outputting in a second wavelength band (e.g. green,red).

A variety of fluorescent stains are available emitting across thevisible from ultraviolet, to blue, green, orange and red. It will beunderstood that the proposed design can be readily modified to use withany desired fluorescent stain/protein with suitable adaptation of theoptical sources, filters and detector. Specifically, the invention canbe applied to cy3 and cy5 stains available from Amersham Biosciences.Non-fluorescent stains to which the invention can be applied includetrypan blue.

It will also be understood that although the term light emitting diodeis used commonly in the art to describe only one type of light sourcebased on diode emission, the term light emitting diodes is to beconstrued broadly in the claims of the present document to cover allforms of light emitting diode sources, including diode lasers, such assemiconductor diode lasers, and superluminescent diodes.

Ways in which a colony of interest can be identified using the apparatusare now described in more detail.

The automated process described herein may used to sort, pick oridentify one or more cell colonies of interest based upon contrastimaging.

Therefore, the present invention may be used for identifying andanalyzing biological molecules that may be present in a cell colony,including, for example, peptides, polypeptides, nucleic acids, andglycosylated and unglycosylated proteins (oligosaccharides, lipids andthe like).

As will be appreciated by a person skilled in the art, a biologicalmolecule, such as a protein of interest, may be detected in a variety ofways. By way of example only, the biological molecule may be detected asfollows:

-   -   1. The expression system that is used to express the protein of        interest may be one which causes the protein of interest to be        secreted from the cell colony into the surrounding medium. If        the expressed protein is secreted into the culture medium, then        typically the expression vector will contain a suitable signal        sequence that directs the secretion of the protein to the        culture medium. A fluorescent antibody, for example, that is        specific for the protein of interest may then be used for the        subsequent identification of cell colonies expressing the        protein of interest.    -   2. The expression system that is used to express the protein of        interest may be one which results in the expression of the        protein of interest on the surface of a cell—such as the cell        membrane. As will be understood by those of skill in the art,        vectors—such as expression vectors—containing coding sequences        may be designed with signal sequences which direct secretion of        the coding sequences through a particular cell membrane. A        fluorescent antibody, for example, may then be used for the        subsequent identification of cell colonies expressing the        protein of interest.    -   3. The cell colony could be made permeable using a cell        permeablization agent, such that a fluorescent antibody for        example, can enter the cell colony and associate with the        protein of interest, whilst still maintaining the viability of        the cell colony.    -   4. The cell colony may comprise an expression vector in which a        protein of interest is fused to a reporter or a ‘tag’—such as        GFP. In this manner, expression of the protein of interest also        results in the expression of the reporter which provides for the        identification of the cell colony. The unique tag sequence is        added to the nucleotide sequence encoding the protein by        recombinant DNA techniques, creating a protein that can be        recognized by an antibody specific for the tag peptide, for        example. A wide variety of reporters may be used in accordance        with the present invention with preferred reporters providing        conveniently detectable signals (e.g. by fluorescence). By way        of example, a reporter gene may encode an enzyme which catalyses        a reaction, which alters light absorption properties. Examples        of reporter molecules include but are not limited to        β-galactosidase, invertase, green fluorescent protein,        luciferase, chloramphenicol, acetyltransferase, β-glucuronidase,        exo-glucanase and glucoamylase. For example, fluorescently        labeled biomolecules specifically synthesized with particular        chemical properties of binding or association may be used as        fluorescent reporter molecules. Fluorescently labeled antibodies        are particularly useful reporter molecules due to their high        degree of specificity for attaching to a single molecular target        in a mixture of molecules as complex as a cell, tissue or        extract of either.

A wide variety of ways to measure fluorescence are available. Forexample, some fluorescent reporter molecules exhibit a change inexcitation or emission spectra, some exhibit resonance energy transferwhere one fluorescent reporter loses fluorescence, while a second gainsin fluorescence, some exhibit a loss (quenching) or appearance offluorescence, while some report rotational movements. Multispectralimaging could also be used.

Applications Examples

Some specific examples of how the apparatus of the invention may be usedare now given.

Selection of Cells for Biopharmaceutical Production

The present invention may also be suited to the automated sorting ofcell colonies which express or secrete enhanced levels of a protein ofinterest. In a preferred embodiment, the protein of interest is abiopharmaceutical protein, such as protein that is useful in thetreatment or diagnosis of disease.

Such cells may be detected according to, for example, the brightness ofthe fluorescence of the cell colony which will correlate with the amountof protein that is expressed. The brightest cell colonies may then bepicked for further analysis/processing.

As described above, the present invention might also be suited to therecovery of cell colonies producing membrane and secreted proteins.

Stem Cells

Differentiation is a process whereby structures and functions of cellsare progressively committed to give rise to more specialized cells.Therefore, as the cells become more committed, they become morespecialized. In the majority of mammalian cell types, celldifferentiation is a one-way process leading ultimately to terminallydifferentiated cells. However, although some cell types persistthroughout life without dividing and without being replaced, many celltypes do continue to divide during the lifetime of the organism andundergo renewal. This may be by simple division or, as in the case ofcells—such as haematopoietic cells and epidermal cells—by division ofrelatively undifferentiated stem cells followed by commitment of one ofthe daughter cells to a program of subsequent irreversibledifferentiation.

Since the present invention is particularly suited for theidentification of cells based upon differences in cell size, shapeand/or replication rate, this may be used to identity and/or isolate oneor more stem cells once they have differentiated into a given cell type.

RNAi

Post-transcriptional gene silencing (PTGS) mediated by double-strandedRNA (dsRNA) is a conserved cellular defense mechanism for controllingthe expression of foreign genes. It is thought that the randomintegration of elements such as transposons or viruses causes theexpression of dsRNA, which activates sequence-specific degradation ofhomologous single-stranded mRNA or viral genomic RNA. The silencingeffect is known as RNA interference (RNAi). The mechanism of RNAiinvolves the processing of long dsRNAs into duplexes of 21-25 nucleotide(nt) RNAs. These products are called small interfering or silencing RNAs(siRNAs) which are the sequence-specific mediators of mRNA degradation.

In differentiated mammalian cells dsRNA >30 bp has been found toactivate the interferon response leading to shut-down of proteinsynthesis and non-specific mRNA degradation. However this response canbe bypassed by using 21 nt siRNA duplexes allowing gene function to beanalyzed in cultured mammalian cells.

In mammals, RNAi can be triggered by delivering either short dsRNAmolecules (siRNAs) directly into the cell, or by delivering DNAconstructs that produce the dsRNA within the cell.

The process and apparatus can be used to identify and pick cells with analtered phenotype for further analysis, since the RNAi inducesmorphological changes in the cells.

Cell Transformation Assays

Differences between transformed and non transformed cells may also bedetected in accordance with the present invention.

Such assays may be used to assay for the transforming abilities ofviruses or chemicals, for example.

Such assays also represent a method for assaying changes consistent withtumorigenesis without knowing the genetic nature of the damage givingrise to the change. After plating at low density, transformed cells,which have an altered phenotype, may be identified using morphologicalcriteria, which is identifiable in an automated process using theimaging capability of the apparatus of the invention.

Cell transformation assays therefore test for genetic alteration thatgives rise to an altered phenotype that is readily identified by themorphological appearance of the transformed cells.

Recovery of Viruses

The presence of a virus often gives rise to morphological changes in ahost cell. Any detectable changes in the host cell due to infection areknown as a cytopathic effect. Cytopathic effects may consist of cellrounding, disorientation, swelling or shrinking, death, detachment fromthe surface, etc.

The cytopathic effects produced by different viruses depend on the virusand the cells on which it is grown. This can be used in the clinicalvirology laboratory to aid in identification of a virus isolate.

Such cytopathic effects may alter the morphology of the cell which canbe detected using the automated process described herein.

Transfection of Genes

In general, the insertion of a gene into a cell requires a dominantselective marker, such as neomycin resistance. The high efficiency of anautomated system described here could make such a marker redundant ascells could be plated at limiting dilutions and colonies picked forfurther analysis.

Immortalization of Cells

Transformation with chemicals or viruses can induce an immortalphenotype. The apparatus and process described herein will allow suchcells to be selected.

Selection of Cell Clones Producing Post Translational Modified Proteins

Therapeutic proteins, such a erythroprotein or tissue plasminogenactivator, owe their serum half lives to sugar residues on the protein.Clones of cells producing a desired modification can be detected with alabeled lectin and then picked.

It will be appreciated that although particular embodiments of theinvention have been described, many modifications/additions and/orsubstitutions may be made within the spirit and scope of the presentinvention.

REFERENCES

-   [1] U.S. Pat. No. 6,198,107: Clare Chemical Research, Inc.-   [2] JP 07-260742: Sanyo Electric Co Ltd.-   [3] WO 98/23950: Oxford Glycosciences (UK) Ltd.-   [4] U.S. Pat. No. 5,587,062: Shimadzu Corporation-   [5] WO 99/051977: Max-Planck-Gesellschaft zur Förderung der    Wissenschaften-   [6] U.S. Pat. No. 5,534,386: Physical Optics Corporation

1. A method for automated picking of animal cell colonies using an apparatus, comprising: a) providing a picking head comprising at least one hollow pin having an inside diameter suitable for picking animal cell colonies of a size that is smaller than said inside diameter, the picking head being movable about the apparatus using positioning motors; b) placing onto the apparatus a sample container having a base on which a plurality animal cell colonies held in a medium are grown, wherein said animal cell colonies have a size smaller than said inside diameter, and also a dispensing container; c) using machine vision and image processing to identify animal cell colony locations in the sample container; d) aligning the picking head with the sample container; e) picking an animal cell colony by aligning one of the hollow pins with one of the animal cell colony locations, moving said one of the hollow pins to a colony picking position in which a distal end of the hollow pin is immersed in the medium over the animal cell colony, and aspirating the animal cell colony into the hollow pin while the distal end of the hollow pin is held in said colony picking position; and f) dispensing the picked animal cell colony by moving the picking head to above the dispensing container and expelling the picked animal cell colony into the dispensing container.
 2. The method of claim 1, wherein the picking step comprises repeating the aligning and aspirating steps for the at least one hollow pin to pick multiple ones of the animal cell colonies.
 3. The method of claim 1, wherein the dispensing container comprises an array of wells separated by a characteristic spacing and the at least one hollow pin comprises a plurality of hollow pins that are also arranged with the characteristic spacing so that the expelling step can be performed in parallel for all the hollow pins.
 4. The method of claim 1, wherein the animal cell colonies are adhered to the sample container and immersed in the medium, and wherein after the introducing step the distal end of the pin is agitated relative to the sample container so as to produce turbulence in the medium to detach the animal cell colony at that location prior to performing the aspirating step.
 5. The method of claim 1, wherein the animal cell colonies are stained with a contrast enhancing agent to assist the image processing.
 6. The method of claim 1, wherein the animal cell colonies are stained with a fluorescent agent to assist the image processing.
 7. The method according to claim 1, wherein the plurality of animal cell colonies comprise or express a biological molecule of interest.
 8. The method of claim 7, wherein the biological molecule of interest is selected from the group consisting of: a peptide, a polypeptide, a nucleic acid, or a glycosylated or unglycosylated protein.
 9. The method according to claim 8 wherein the protein of interest is a biopharmaceutical protein.
 10. The method of claim 1, wherein the animal cell colonies are held suspended in the medium.
 11. The method of claim 1, wherein the animal cell colony comprises a plurality of cells.
 12. The method of claim 1, wherein the animal cell colony consists of a single cell.
 13. The method of claim 1, wherein the distal end of the hollow pin is offset from the base of the sample container by an offset distance, and the offset distance between the distal end of the hollow pin and the base of the sample container is in a range between about 0.1 mm and about 0.4 mm.
 14. The method of claim 13, wherein the offset distance between the distal end of the hollow pin and the base of the sample container is from about 0.25 mm to about 1.0 mm.
 15. An apparatus for picking animal cell colonies comprising: an apparatus bed useful for arranging a sample container comprising a base on which a plurality of animal cell colonies held in a medium are grown; a camera useful for capturing images of the animal cell colonies; a computer for controlling the apparatus; control software and image processing software resident in the computer, wherein the image processing software is useful for identifying animal cell colony locations from captured images, and the control software is useful for controlling the picking of animal cell colonies through interaction with the image processing software, and control of various components of the apparatus; and a picking head movable relative to the apparatus bed under control of the control software and using positioning motors to animal cell colony locations identified by the image processing software from said images, wherein the picking head comprises at least one hollow pin, the at least one hollow pin comprising an inner pin and an outer pin, wherein the inner pin is recessed axially inside an end of the outer pin, wherein the inner pin has an inside diameter, and wherein the animal cell colonies being picked have a size that is smaller than inside diameter of the inner pin, the at least one hollow pin being connected to a drive that introduces a distal end of the at least one hollow pin into the sample container, and further connected through respective at least one fluid conduct to a pressure controller that is operable to aspirate quantities of the medium from the sample container into the inner pin of the at least one hollow pin, to retain the medium and to expel it when required, thereby allowing animal cell colonies to be picked from the medium by the at least one hollow pin, and wherein the control software of the computer is operable to control the apparatus for colony picking by: (i) capturing an image of the animal cell colony with the camera; (ii) performing image analysis with the image processing software to detect those animal cell colonies to pick, thereby creating a pick list of target colonies; (iii) assigning the apparatus to collect the target colonies from the pick list with the control software, wherein the target colonies are picked by repeatedly performing the following actions specified by the control software: (a) aligning one of the at least one hollow pin to an animal cell colony location in the pick list; (b) moving said hollow pin to a colony picking position in which a distal end of the hollow pin is immersed in the medium over the animal cell colony, and (c) aspirating the animal cell colony at that location into the inner pin while the distal end of the hollow pin is held in said colony picking position.
 16. The apparatus of claim 15, wherein the apparatus further comprises a drive mechanism useful for causing lateral oscillation of distal ends of the at least one hollow pin to facilitate detachment of animal cell colonies adhered to the sample container.
 17. The apparatus of claim 16, wherein the drive mechanism is configured to cause rotary motion of the distal ends of the at least one hollow pin.
 18. The apparatus of claim 15, wherein the at least one hollow pin comprises a plurality of hollow pins that are arranged in a characteristic spacing matched to a well plate standard spacing.
 19. A method that comprises using the apparatus according to claim 15 for identifying an animal cell colony comprising or expressing a biological molecule of interest.
 20. The method according to claim 19, wherein the biological molecule of interest is selected from the group consisting of: a peptide, a polypeptide, a nucleic acid, or a glycosylated or unglycosylated protein.
 21. The method according to claim 19, wherein the glycosylated or unglycosylated protein of interest is a biological molecule.
 22. A method that comprises using the apparatus according to claim 15 for picking animal cell colonies held suspended in a medium, wherein the method comprises the steps of picking an animal cell colony by aligning the at least one hollow pin with one of the animal cell colony locations, moving the at least one hollow pin to a colony picking position in which a distal end of the at least one hollow pin is immersed in the medium over the animal cell colony, and aspirating the animal cell colony into the at least one hollow pin while the distal end of the at least one hollow pin is held in said colony picking position.
 23. A method that comprises using the apparatus according to claim 15 for identifying and picking an animal cell colony comprising a plurality of cells, wherein the method comprises the steps of picking an animal cell colony by aligning the at least one hollow pin with one of the animal cell colony locations, moving the at least one hollow pin to a colony picking position in which a distal end of the at least one hollow pin is immersed in the medium over the animal cell colony, and aspirating the animal cell colony into the at least one hollow pin while the distal end of the at least one hollow pin is held in said colony picking position.
 24. A method that comprises using the apparatus according to claim 15 for identifying and picking an animal cell colony consisting of a single cell, wherein the method comprises the steps of picking an animal cell colony by aligning the at least one hollow pin with one of the animal cell colony locations, moving the at least one hollow pin to a colony picking position in which a distal end of the at least one hollow pin is immersed in the medium over the animal cell colony, and aspirating the animal cell colony into the at least one hollow pin while the distal end of the at least one hollow pin is held in said colony picking position.
 25. The apparatus of claim 15, wherein the at least one hollow pin comprises a plurality of hollow pins.
 26. The apparatus of claim 16, wherein the drive mechanism is arranged in the picking head.
 27. The apparatus of claim 16, wherein the drive mechanism is not provided by the positioning motors.
 28. The apparatus of claim 15, wherein the distal end of the hollow pin is offset from the base of the sample container by an offset distance while in said colony picking position, and the offset distance between the distal end of the hollow pin and the base of the sample container is in a range between about 0.1 mm and about 4.0 mm.
 29. The apparatus of claim 28, wherein the offset distance between the distal end of the hollow pin and the base of the sample container is from about 0.25 mm to about 1.0 mm. 